Virus-based expression systems can be used for rapid protein production in plants (for review see: Porta & Lomonossoff, 1996, Mol. Biotechnol., 5, 209-221; Yusibov et al., 1999, Curr. Top. Microbiol. Immunol, 240, 81-94) and are a powerful tool for functional genomics studies (Dalmay et al., 2000, Plant Cell, 12, 369-379; Ratcliff et al., 2001, Plant J., 25, 237-245; Escobar et al., 2003, Plant Cell, 15, 1507-1523). Numerous publications and patents in the field describe systems based on DNA and RNA viral vectors (Kumagai et al., 1994, Proc. Natl. Acad. Sci. USA, 90, 427-430; Mallory et al., 2002, Nature Biotechnol. 20, 622-625; Mor et al., 2003, Biotechnol. Bioeng., 81; 430-437; U.S. Pat. Nos. 5,316,931; 5,589,367; 5,866,785; 5,491,076; 5,977,438; 5,981,236; WO02088369; WO02097080; WO9854342). The existing viral vector systems are usually restricted to a narrow host range in terms of their best performance and even the expression level of such vectors in their most favourable host is far below the upper biological limits of the system.
RNA viruses are the most suitable for use as expression vectors, as they offer a higher expression level compared to DNA viruses. There are several published patents which describe viral vectors suitable for systemic expression of transgenic material in plants (U.S. Pat. Nos. 5,316,931; 5,589,367; 5,866,785). In general, these vectors can express a foreign gene as a translational fusion with a viral protein (U.S. Pat. Nos. 5,491,076; 5,977,438), from an additional subgenomic promoter (U.S. Pat. Nos. 5,466,788; 5,670,353; 5,866,785), or from polycistronic viral RNA using IRES elements for independent protein translation (WO0229068). The first approach—translational fusion of a recombinant protein with a viral structural protein (Hamamoto et al., 1993, BioTechnology, 11, 930-932; Gopinath et al., 2000, Virology, 267, 159-173; JP6169789; U.S. Pat. No. 5,977,438) gives significant yield. However, the use of such an approach is limited, as the recombinant protein cannot be easily separated from the viral one. One of the versions of this approach employs the translational fusion via a peptide sequence recognized by a viral site-specific protease or via a catalytic peptide (Dolja et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10208-10212; Gopinath et al., 2000, Virology, 267, 159-173; U.S. Pat. Nos. 5,162,601; 5,766,885; 5,491,076).
Expression processes utilizing viral vectors built on heterologous subgenomic promoters provide a good level of protein production (U.S. Pat. No. 5,316,931). The most serious disadvantage of such vectors and many others is their limited capacity with regard to the size of DNA to be amplified. Usually, stable constructs accommodate inserts of not more than one kb. In some areas of plant functional genomics this may not be such a serious limitation as G. della-Cioppa et al. (WO993651) described the use of TMV-based viral vectors to express plant cDNA libraries with the purpose of silencing endogenous genes. Additionally, as such vectors are capable of systemic movement and produce coat protein, significant resources of the plant are diverted from the synthesis of recombinant protein. The low expression levels achieved so far with such plant viral expression systems are a major reason why these systems are hardly competitive with other expression systems like bacterial, fungal, or insect cell expression systems. Low expression levels give rise to very high downstream costs for protein isolation and purification in a huge background of plant material. Therefore, costs for downstream processing quickly decrease, as the yield of the protein or product of interest per unit plant biomass increases. Also, a biological safety of such vectors are an issue, as they are able to form infectious viral particles.
An alternative two-component system requiring a helper virus was developed by Turpen (U.S. Pat. No. 5,811,653; U.S. Pat. No. 5,889,191; U.S. Pat. No. 5,965,794); this approach relies on a system of a virus and a helper virus, whereby the helper virus provides a replicase function, whereas the main replicon is deficient in replicase activity. This system is not practical because viral RNA-dependent RNA polymerase (replicase) works inefficiently with substrate RNAs provided in trans. A possible explanation of such inefficiency is that TMV RNA-dependent RNA polymerase is a heterodimer consisting of a 126 kDa protein and a 183 kDa read-through protein (Watanabe et al., 1999, J. Virol. 73, 2633-2640). It was shown that at least one component of this heterodimer, the 126 kDa protein, appeared to work primarily in cis (Lewandowsky & Dawson, 2000, Virology, 271, 90-98). There are several publications concerning the complementation in trans of other viral functions, like cell-to cell and systemic movement. The MP and CP can be provided in trans either by a transgenic host or by another virus. For example, mutants of TMV with frameshifts within the MP or CP gene were unable to locally or systemically infect inoculated tobacco plants, but acquired the lost functions in transgenic tobacco plants expressing the wild-type MP or CP gene (Holt & Beachy, 1991, Virology, 181, 109-117; Osbourn, Sarkar & Wilson, 1990, Virology, 179, 921-925). These works did not address the issue of creating virus-based vectors for expressing a heterologous sequence of interest, but rather studied the biological functions of different viral proteins. Another work describes the complementation of long distance movement of a CP-deficient TMV expressing GFP by a chimeric TMV carrying ORF3 of groundnut rosette umbravirus (GRV) (Ryabov, Robinson & Taliansky, 1999, Proc. Natl. Acad. Sci. USA, 96, 1212-12170). However, as it follows from the results, the efficiency of GFP expression in systemic leaves of plants co-infected with CP-deficient TMV expressing GFP and TMV having CP replaced by ORF3 of GRV was significantly lower than in plants infected with systemic TMV expressing GFP. It appears that this low expression level may be due to the presence and competition of both viral vectors in systemic leaves. Moreover, all experiments mentioned above led to the formation of infectious viral particles in systemic leaves and are therefore not acceptable for use in the environment from the point of view of biological safety.
Another system proposed by C. Masuta et al. (U.S. Pat. No. 5,304,731) proposes to use a satellite CMV RNA virus to be used as a carrier of the heterologous sequence of interest, and a helper virus that provides functions necessary for CMV RNA replication. To the best of our knowledge, the system is highly inefficient.
A serious concern with prior art virus-based plant expression systems is biological safety. On the one hand, high infectivity of the recombinant virus is highly desired in order to facilitate spread of the virus throughout the plant and to neighbouring plants, thereby increasing the yield of the desired gene product. On the other hand, such a high infectivity compromises containment of the recombinant material since spread to undesired plants can easily occur. Consequently, safer virus-based plant expression systems are highly desired.
There is presently no biologically safe large-scale transgene expression system built on plant RNA viral vectors capable of moving systemically and providing for the yield and efficiency required for technical applications. The existing systemic vectors suffer from a low yield of recombinant product.
Therefore, it is an object of this invention to provide an environmentally safe plant viral expression system for high-yield production of a protein of interest. It is another object of the invention to provide a process of replicating and/or expressing a nucleotide sequence of interest in a plant or plant part, which is of improved ecological and biological safety. It is another object to provide a process of protein production in plants having efficiency enabling competitive large-scale protein production in plants.